Summary Sepsis is now the leading cause of death in US hospitals and there are currently no effective pharmacological treatments for sepsis. Stromal cell-based therapies have shown efficacy in treating sepsis in experimental models and have been approved for use in multiple countries for various immune dysregulation diseases. Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells existing in all lymphoid organs including fat associated lymphoid cluster (FALC) in adipose tissue. We have shown that TLR9 signaling suppresses chemokine production in FRCs. Adoptive transfer of Tlr9-deficient FRCs decreased mortality, bacterial load, and systemic inflammation compared with wild type (WT) FRCs after cecal ligation and puncture (CLP). Here, we identified two distinct subsets of FRCs (Ly6Chi and Ly6Clo) in mesenteric FALCs at baseline and after CLP. Importantly, the Ly6Chi FRC subset express similar top marker genes as the CD55+ stromal cell subset that was previously described in both mouse and human subcutaneous adipose tissue. Ly6Chi FRCs are enriched in the innate immune response-related genes after CLP, whereas Ly6Clo FRCs are enriched in the humoral immune response-related genes. Furthermore, we found that both Ly6Chi and Ly6Clo FRCs from Tlr9-/- mice increased gene expression associated with inflammation, proliferation, and extracellular matrix remodeling compared with WT FRCs at baseline and after CLP. Based on these findings, we hypothesize that TLR9 plays critical roles in regulating the biology of distinct FRC subsets, and that modulation of TLR9 signaling in a subset-specific manner may improve the efficacy of FRC-based therapy in sepsis. We will test our hypothesis by pursuing two specific aims: Aim 1: To determine the mechanisms of TLR9-mediated regulation of FRC biology in mouse and human adipose FRC subsets. We will take advantage of single cell technologies (single cell RNA-sequencing and Mass cytometry) combined with the FRC-specific Tlr9-/- mice to determine the role of TLR9 in gene expression, cell fate, and immunoregulatory functions in individual FRC subset in vivo and in vitro. We will also validate the findings from mouse FRCs in human adipose FRCs. Aim 2: To determine the impact of TLR9 inhibition preconditioning on FRC therapy in intra-abdominal sepsis. We will determine the therapeutic efficacy of TLR9 inhibition preconditioned FRC subsets in two clinically relevant intra-abdominal sepsis models: (1) CLP-induced polymicrobial peritonitis; and (2) intra-abdominal infection of a human strain of Escherichia coli. We will also use an LPS-induced peritonitis model to determine the mechanisms underlying the beneficial effects of FRC-based therapy and the impact of TLR9 on individual FRC subset therapies. Our study will advance understanding of the diversity and biology of FRC subsets as well as the regulation of TLR9 in these distinct subsets, which will discover new strategies to modify selective FRC subsets to improve efficacy of FRC-based therapy for sepsis and other immune dysregulation diseases.